Hydrogen sulfide production during early yeast fermentation correlates with volatile sulfur compound biogenesis but not thiol release

Abstract Yeasts undergo intensive metabolic changes during the early stages of fermentation. Previous reports suggest the early production of hydrogen sulfide (H2S) is associated with the release of a range of volatile sulfur compounds (VSCs), as well as the production of varietal thiol compounds 3-sulfanylhexan-1-ol (3SH) and 3-sulfanylhexyl acetate (3SHA) from six-carbon precursors, including (E)-hex-2-enal. In this study, we investigated the early H2S potential, VSCs/thiol output, and precursor metabolism of 11 commonly used laboratory and commercial Saccharomyces cerevisiae strains in chemically defined synthetic grape medium (SGM) within 12 h after inoculation. Considerable variability in early H2S potential was observed among the strains surveyed. Chemical profiling suggested that early H2S production correlates with the production of dimethyl disulfide, 2-mercaptoethanol, and diethyl sulfide, but not with 3SH or 3SHA. All strains were capable of metabolizing (E)-hex-2-enal, while the F15 strain showed significantly higher residue at 12 h. Early production of 3SH, but not 3SHA, can be detected in the presence of exogenous (E)-hex-2-enal and H2S. Therefore, the natural variability of early yeast H2S production contributes to the early output of selected VSCs, but the threshold of which is likely not high enough to contribute substantially to free varietal thiols in SGM.


Introduction
Hydrogen sulfide (H 2 S) produced by yeast has been suggested to play important roles in heavy metal detoxification (Kikuchi 1965, Ono et al. 1991 ), population sync hr on y (Sohn et al. 2000, Kwak et al. 2003 ), and c hr onological longe vity (Hine et al. 2015 ).Yeastderived H 2 S is mainly produced from exogenous sulfate via the sulfur assimilation pathway (SAP) in response to the metabolic r equir ement for sulfur-containing amino acids (Linderholm et al. 2008, Rose et al. 2017 ), cysteine catabolism (Stipanuk 2004 ), and from the application of elemental sulfur under an anaerobic and low-pH envir onment (Ar aujo et al. 2017 ).Of note, H 2 S has been suggested to play metabolic and pr otectiv e r oles in the cell during the early phase of fermentative transition, where a time-critical nutritional switch and oxidative stress response occur (Jiranek et al. 1995, Kwak et al. 2003 ).
While commonl y consider ed to be an undesir able off-odor in wines, earl y pr oduction of H 2 S has been linked to the accumulation of thiol compounds, specificall y 3-sulfan ylhexan-1-ol (3SH) and its acetate ester deri vati ve, 3-sulfanylhexyl acetate (3SHA), in the presence of plant-derived α,β-unsaturated aldehydes and alcohols, via direct addition to the double bond (Schneider et al. 2006, Harsch et al. 2013 ).Ho w ever, the contribution of this pathway to total thiol output is disputed (Schneider et al. 2006, Subileau et al. 2008, Roland et al. 2010, Harsch et al. 2013, Bon-naffoux et al. 2018 ).Inter estingl y, the concentr ations of gr a pederiv ed α,β-unsatur ated six-carbon pr ecursors, suc h as ( E )-hex-2enal and its deri vati ves, rapidly diminish within a few hours of yeast inoculation (Harsch et al. 2013 ), potentially due to active yeast detoxification (Trotter et al. 2006 ).This pr ocess likel y occurs before inoculation in naturally harvested and pressed grape must, due to interactions with indigenous communal microorganisms (Joslin andOugh 1978 , Hammerbacher et al. 2019 ).Alternativ el y, fr ee ( E )-hex-2-enal, like other α,β-unsaturated aldehydes , can be sca v enged by plant-deriv ed glutathione S-tr ansfer ase, or detoxified by plant-derived enzymes including aldehyde dehydr ogenase, aldo-keto r eductase, NADPH-de pendent 2-alk enal reductase, and alkenal/one oxidoreductase, albeit in a relatively minor and slo w er process (Mano et al. 2002, 2019, Yamauchi et al. 2011, Mano 2012 ).The window of precursor -H 2 S concurr ence is, ther efor e, consider ed limited to the immediate-early phase of the fermentation ( < 12 h), when H 2 S production begins to incr ease.Mor eov er, we pr e viousl y observ ed the accumulation of a range of volatile sulfur compounds (VSCs), concurrent with H 2 S production, during the course of laboratory fermentation in synthetic gr a pe medium (SGM) (Kinzurik et al. 2016 ).Specificall y, c hemical pr ofiling of finished ferments pr oduced from full-length fermentations revealed that the levels of ethanethiol, S -ethyl acetate, and diethyl disulfide in the final wine were associated with H 2 S production.Tracing of isotope incorporation further suggested that these compounds are direct downstr eam pr oducts of H 2 S (Kinzurik et al. 2016 ).Mor eov er, exogenous H 2 S spiking into activ el y fermenting yeast suggested yeastmediated production of specific downstream VSCs (Kinzurik et al. 2020 ).
T he abo ve findings highlight the need to further understand the role of H 2 S during the early stage of yeast fermentation, where a dynamic physiological transition takes place inside yeast cells.Indeed, it is well documented in pr e vious r eports that remarkable inter-str ain v ariabilities in H 2 S potential ar e observ ed among Sacc harom yces cerevisiae yeasts, including natural isolates, commercial strains, and laboratory deletants (Kumar et al. 2010, Winter et al. 2014 ).Ho w e v er, suc h r eports ar e usuall y insufficient in terms of displaying detailed temporal resolution of H 2 S biogenesis, nor do they attempt to associate H 2 S e v aluation with quantitative c hemical pr ofiling during fermentation.Although we have qualitativ el y established the association between H 2 S with its immediate downstream products (Kinzurik et al. 2015 ), this previous work did not differentiate the impact of H 2 S on VSC production at the earl y sta ge of fermentation fr om finished wine samples due to the technical limitations of silver nitrate tester tubes.Given this appr oac h dir ectl y quantifies the liber ated H 2 S pool, it ther efor e can only detect and quantify H 2 S after ∼18 h of fermentation.
In this w ork, w e aimed to couple the earl y sta ge (12 h) H 2 S profiles of a panel of laboratory reference and commonly used commer cial wine y east str ains, measur ed via methylene blue reduction assay, alongside VSC and volatile thiol quantification in model juice using gas c hr omatogr a phy-mass spectr ometry (GC-MS) and liquid c hr omatogr a phy-mass spectr ometry (LC-MS), in an attempt to c har acterize the earl y H 2 S-VSC-thiol pr ofile.Furthermor e, the r elationships among earl y H 2 S pr oduction, downstr eam VSC formation, and final thiol output contributed from a C 6 precursor, specifically ( E )-hex-2-enal, were assessed for potential applications in yeast phenotype screening for the wine industry.

Media and reagents
All chemicals, unless otherwise indicated, were purchased from Sigma-Aldric h (German y).Anal ytical-gr ade anhydr ous ethanol w as pur c hased fr om Ajax Finec hem (Tar en Point, NSW, Australia), and D-glucose w as pur chased from Merck (Kenilworth, NJ, USA).The internal standard dimethyl-d 6 sulfide ( d 6 -DMS), along with methionol, methanethiol, and dimethyl disulfide, were purc hased fr om Sigma-Aldric h (Darmstadt, German y).Liquid YPD or YPD agar plates were used for routine maintenance of standard yeast cultur es, while c hemicall y defined SGM mimic king gr a pe juice (21 • Brix, pH 3.2, yeast assimilable nitrogen 300 mg L −1 ) was used for fermentativ e gr owth, as described in pr e vious r eports (Kinzurik et al. 2015, Deed et al. 2019 ).To cater for the methionine auxotrophy of the BY4741 strain, 10-fold concentrations of Lmethionine were supplemented in the final SGM (0.3 mM).( E )-hex-2-enal (Sigma-Aldrich, Darmstadt, Germany) stock solution was fr eshl y pr epar ed and aliquoted by dissolving in anhydr ous ethanol at 1.5 mg mL −1 and supplemented into the medium pr epar ation at a final concentration of 1.5 mg L −1 just before yeast seeding.YNB medium (yeast nitrogen base without amino acids or ammonium sulfate 1.7 g L −1 , ammonium chloride 0.3 g L −1 , L-malic acid 3 g L −1 , citric acid 0.2 g L −1 , sucrose 200 g L −1 , pH = 3.2) was used for early precursor kinetics.

Methylene blue reduction based H 2 S detection
Methylene blue-based (MetB) H 2 S detection in live yeast cultures was performed according to pr e vious r eports (Winter andCurtin 2012 , Winter et al. 2014 ) with minor modifications.Briefly, stationary-phase yeast pr ecultur es wer e pr epar ed with ov ernight incubation at 28 • C in liquid YPD.In each well of a 96-well microtiter plate (Corning, USA), a 170 μl aliquot of SGM, 20 μl aliquot of a MetB reaction mix (0.5 mg mL −1 MetB, 50 mM citric acid buffer at pH 4.5), and 10 μl of yeast culture were mixed thoroughly to a final cell count of 2 × 10 6 cell mL −1 .Assays were carried out in quadruplicate with uninoculated SGM as the blank control.The microtiter plate was placed in a SpectraMax iD3 plate reader (Molecular Devices , C A, USA) with intermittent shaking (10 s at intermediate intensity for e v ery 10 min) at 25 • C. Absorbances at 663 and 600 nm wer e r ecorded automaticall y at 10-min interv als for 12 h.Cell gr owth contr ols wer e included by r ecording the optical densities at 600 nm concurr entl y for cells without the MetB detection mix.

Labor a tory fermenta tion
Laboratory-scale fermentation was performed as described previously (Deed et al. 2019 ).Briefly, precultures of each yeast strain wer e pr epar ed ov ernight.Yeast cells wer e collected by centrifugation at 3000 g for 5 min, then washed with sterile water.Starter cultur es wer e seeded at 2 × 10 6 cells mL −1 in triplicate into 250 ml Erlenmeyer flasks sealed with water-filled airloc ks, eac h contain-  ing 100 ml of SGM and supplemented with 1.5 mg L −1 of ( E )-hex-2enal.After 12 h of incubation at 25 • C with 120 rpm agitation, fermentations were harvested by centrifugation at 3000 g for 15 min.Cell-fr ee supernatants, r epr esenting the finished wines, wer e collected and stored in pol ypr opylene 70-ml plastic containers at −80 • C prior to chemical profiling.
For the early precursor kinetics experiment, overnight precultures of BY4741 and BY4741-oye2 oye3 inoculants were seeded into YNB medium spiked with 1.5 mg L −1 of ( E )-hex-2-enal at 2 × 10 6 cells mL −1 final cell density.NaSH was supplemented just before yeast inoculation at a final concentration of 10 mg L −1 as the exogenous H 2 S donor.Samples wer e harv ested at 1, 2, 3, 6, and 24 h of fermentation by centrifugation at 3000 g for 15 min.Two initial samples for each fermentation run were also collected be-fore (0 h medium only) and immediately after yeast inoculation (0 h).

VSC quantification via HS-SPME/GC-MS
VSCs in finished wines wer e extr acted using the method developed and emplo y ed in pr e vious r eports (Nguyen et al. 2012, Kinzurik et al. 2015, Deed et al. 2019 ).Defrosted wine sample (10 ml) was saturated with magnesium sulfate heptahydrate (2.6 g) and purged with nitrogen to prevent analyte degradation, follo w ed b y injection of 50 μl of an internal standard mix (30 μg L −1 d 6 -DMS, 2 μg L −1 DPDS, 547 μg L −1 3-methytlthio-1hexanol). Head space solid phase micr o-extr action coupled with gas c hr omatogr a phy-mass spectr ometry (HS-SPME/GC-MS) on an Ag ilent Technolog ies 7890 GC system coupled with a 5975C in-

Da ta anal ysis
The cum ulativ e H 2 S pr ofile of eac h str ain was estimated as described in pr e vious r eports (Winter and Curtin 2012 ).GC-MS sample analysis was performed using the MassHunter workstation softwar e (v ersion B.07.01, Agilent, Santa Clara, CA, USA).Correlations between H 2 S production and VSC production from the early fermentation period were tested using Spearman's rank test ( P < .05)and the 95% CI estimated using jackknife Euclidean likelihood-based inference (de Carvalho and Marques 2012 ).Oneway ANOVA with post hoc Tuk e y's HSD test ( P < .05)was performed to analyze strain variability in precursor metabolism.

Results and discussion
This work e v aluates the pr oduction of H 2 S during the earl y sta ge of alcoholic fermentation using a collection of laboratory reference and commer cial y east strains fed with c hemicall y defined SGM (Deed et al. 2019 ).Pr e vious studies hav e shown that earl y H 2 S release in the presence of the short-lived unsaturated C 6 com-pound ( E )-hex-2-enal induces direct, yeast-mediated, formation of varietal thiols, including 3SH/3SHA (Harsch et al. 2013 ).It is therefor e inter esting to e v aluate whether v ariation in S. cerevisiae earl y H 2 S-producing potential is associated with thiol-producing phenotypes in the presence of ( E )-hex-2-enal.

Early H 2 S profiles during fermentation of the labor a tory and commercial strains
We surveyed the early H 2 S production profile of 11 laboratory reference and commonly used commer cial y east strains (two laboratory r efer ence str ains: BY4741 and BY4741, and nine commercial strains: EC1118, F15, M2, MaxiThiol, RM11, UCD522, VIN13, VL3, and X5).Significant variability in the H 2 S-producing potential of the 11 selected strains was demonstrated as early as 12 h after initiation of fermentation (Fig. 1 ) using time-course spectrophotometry with the MetB reduction method.H 2 S released from the ferments was below the detection threshold of conventional H 2 S detector tubes (Kinzurik et al. 2015, Huang et al. 2016 ).Laboratory r efer ence str ains sho w ed lo w er H 2 S accum ulation compar ed with the commercial strains.Of note, BY4741 has pr e viousl y been reported to be a high producer of H 2 S in terms of total H 2 S quantification over the course of a full-length fermentation (Kinzurik et al. 2015 ) compared with its BY4743 diploid counterpart.It is hypothesized that the low accumulation of H 2 S in BY4741 fermentations observed in the current study, and generally in fermentations produced by the laboratory reference strains, arises from lo w er adaptiveness to the fermentative environment (high sugar, low pH) (Spiropoulos et al. 2000, Linderholm et al. 2008 ).Consistent measur ements wer e obtained for pr e viousl y r eported moder ate-high H 2 S producers, including F15 and UCD522 (Mendes-Ferr eir a et al. 2010 , Kinzurik et al. 2016 , Xing andEdw ar ds 2019 ).H 2 S quantifications in pr e vious r eports hav e shown extensiv e v ariation in H 2 S production among a viney ar d y east collection (Spiropoulos et al. 2000, Mendes-Ferr eir a et al. 2002 ), as well as among single gene deletants (Winter et al. 2014 ).In these studies, most of which aimed at identifying low-H 2 S-pr oducing str ains that are favorable in winemaking pr actice, H 2 S pr ofiles ar e usuall y r eported with less tempor al r esolution as an accum ulation within at least 2-3 da ys .In contrast, our data addresses the H 2 S profile at the early transitional phase of fermentation with a snapshot suggesting discernible variabilities before stationary fermentation is plateaued.
The accumulation of several VSCs, including ethanethiol (EtSH), S-ethyl thioacetate (ETA), and diethyl disulfide (DEDS), during a full-length fermentation has pr e viousl y been reported to be the result of H 2 S production (Kinzurik et al. 2016 ). 34S isotope labeling provided evidence of a direct association between H 2 S and these VSCs in the finished wine.H 2 S is thought to react with either acetaldehyde or ethanol to produce EtSH (Rauhut and Kurbel 1994 ), which then dimerizes to produce DEDS in a yeastindependent manner (Bobet et al. 1990 ), as is consistent in this experiment.DMDS is suggested to be a downstream deri vati ve of methanethiol (MeSH).While MeSH itself was observed in the current study to be only marginally correlated with H 2 S production [ ρ = 0.161, 95% CI ( −0.245, 0.566)], MeSH is the direct product of methionine catabolism (Landaud et al. 2008, Deed et al. 2019 ), and is spontaneously oxidized/dimerized into DMDS (Chin andLindsay 1994 , Kreitman et al. 2017 ).Notably, exogenous H 2 S spiking into yeast-containing active fermentation leads to increased MeSH (Kinzurik et al. 2020 ).This can be inter pr eted as an indirect r esult fr om incr eased methionine av ailability via yeast SAP, thus associated with ele v ated MeSH pr oduction via demethiolation in the feedback regulation (Arfi et al. 2002, Perpète et al. 2006 ), which further increase the concentration of DMDS, its immediate downstream oxidant.
The association of ETA with H 2 S production during fermentation was pr e viousl y suggested (Kinzurik et al. 2016 ), whereas a recent study r e v eals that ETA r a pidl y decr eases in the presence of yeast cells (Jiménez-Lorenzo et al. 2022 ).Our data, howe v er, could not establish a positive correlation between H 2 S and ETA.In contr ast, the corr elation of H 2 S with DEDS suggests the dominance of the alternative pathway of the fate of EtSH.Inter estingl y, ther e is evidence of the reverse conversion of DEDS to EtSH (Bobet et al. 1990 ), ETA to EtSH, and DMDS to MeSH occurring in post-bottling finished wine (Bekker et al. 2018 ).
2-ME is proposed to be the production of cysteine Ehrlich degradation (Silv a Ferr eir a et al. 2003, Vermeulen et al. 2006 ).Although a range of reports has accounted for its quantification in distinct wine varieties and beer (Rapp et al. 1985, Vermeulen et al. 2006, Fedrizzi et al. 2007, Jiménez-Lorenzo et al. 2022 ), the association of 2-ME and early H 2 S is documented for the first time .T he early formation of 2-ME, m uc h like DMDS, could be explained by the vigorous yeast metabolism during the immediate-early stage of fermentation, which may exhibit distinct profiles in finished or aging wines, as the decrease of 2-ME concentration over time invariably shown in pr e vious inv estigations (Silv a Ferr eir a et al. 2003, Fedrizzi et al. 2007 ).
Together, these results suggest that the concentration of downstr eam pr oducts corr elates with the efflux of H 2 S at the v ery earl y stage of fermentation, some of which could serve as surrogate indicators when direct measurement of H 2 S is not possible.Ov er all, a distinct volatile metabolite landscape and pathway pr efer ence wer e r e v ealed as compar ed with stationary phase fermentation or further process.Further longitudinal investigation is required along the course of full-scale fermentation to illustrate the dynamic interconversion of volatile compounds via distinct biological/c hemical pr ocesses.
Although the SGM was spiked with ( E) -hex-2-enal prior to fermentation, no quantifiable 3SH or 3SHA was observed in any of the 12 h fermentation samples.We cannot determine whether the absence of these thiols is attributable to the insufficient H 2 S production in the SGM during early fermentation, possibly due to the composition of the SGM, and/or due to thiol production being below the detection limits of the two methods employed.T here ha ve been limited attempts to quantify thiol production using a c hemicall y defined SGM, and none of them, to our knowledge , ha ve in vestigated the very early phase of fermentation.Santiago and Gardner quantified thiols in end-point ferments produced from SGM spiked with cysteinylated and glutathionylated pr ecursors, and r eported a maxim um of 8%-10% of total thiol yield (Santiago and Gardner 2015 ).Jelley et al. quantified endpoint 3SH pr oduced fr om SGM supplemented with gr a pe marc extr act as sources of thiol precursors at 216.2 or 1244.4 ng L −1 according to the le v els of gr a pe marc extr act input (Jelley et al. 2020 ).On the other hand, there have been even fewer attempts to study the time-course evolution of 3SH/3SHA during fermentation, es-pecially in the early phase.Tominaga et al. reported the production of these thiols from the VL3c yeast strain in Sauvignon blanc gr a pe m ust ov er a 5 d fermentation, with the first observation ( ∼550 ng L −1 of 3SH) reported on day 2 after initiation (Tominaga et al. 1998 ).Bonnaffoux et al. also using Sauvignon blanc gr a pe m ust, reported ∼117.5 ng L −1 of 3SH at 15 h after initiation (Bonnaffoux et al. 2018 ).In current w ork, tw o distinct quantification appr oac hes to measure thiols were utilized.A QuEChERS-based extraction yielded very poor recovery of the organic fraction, possibly due to the high sugar le v els in the samples .T her efor e, 3SH and 3SHA were also analyzed using the ETP-SPE extraction method.Ho w e v er, neither thiol could be detected in any sample using either method.Both methods are shown to be efficient for free thiol quantification in a range of alcoholic be v er a ges (Herbst-Johnstone et al. 2013, Tonidandel et al. 2021, Jelley et al. 2022 ), although there is insufficient report of their application in unfermented or lightly fermented "juice"-like media.
It is ther efor e of inter est to inv estigate the effect of H 2 S pr oduction b y y east on early thiol output in real grape juice.In addition to the considerable inter-batch variabilities of n utrients, o xygen le v els, and pr ecursors, gr a pe juice contains natural yeast and nonyeast microbial flora.Although chemical measures can be taken to suppress the growth of some unwanted micr oor ganisms, some natur al yeasts ar e selected for high tolerance of common antimicr obial tr eatments (e .g. SO 2 ).T his ma y confound the deduction of the H 2 S-thiol relationship using a pure starter culture with exogenous ( E )-hex-2-enal precursor, since it will be rapidly metabolized b y y east and other micr oor ganisms.
Mor eov er, an alternativ e pathwa y ma y compete with H 2 S for the precursor ( E )-hex-2-enal in juice and GSH-containing SGM, which may also explain the lack of detectable early 3SH and 3SHA.It is hypothesized that the majority of available ( E )-hex-2-enal could first conjugate with GSH by glutathione S-tr ansfer ase, follo w ed b y conversion to c ystein ylated pr ecursor catal yzed by γglutamyl tr ansfer ase and carbo xype ptidase, before further downstr eam conv ersion by β-l ysis to form 3SH (K oba yashi et al. 2011, Helwi et al. 2016, Thibon et al. 2016 ).Alternativ el y, ( E )-hex-2-enal can conjugate dir ectl y with cysteine to form S -cysteine conjugates (Tominaga et al. 1998, Tominaga and Dubourdieu 2000, Starkenmann 2003, Starkenmann et al. 2008 ).It leads, ho w e v er, to the question of the actual impact of H 2 S dir ectl y on final thiol production, either in the earl y sta ge or over the full course fermentation, as a major fraction of its target ( E )-hex-2-enal would be sequestered by a plethora of nucleophilic attackers, including GSH and cysteine .T his suggests that alternative pathwa ys ar e likel y to predominate since it is estimated that the contribution from direct formation of 3SH/3SHA may be limited to only ∼5% of final thiol output (Subileau et al. 2008, Harsch et al. 2013 ).Nonetheless, the lack of thiol detection in 12 h early fermentations alone could not exclude the possibility that the insufficient H 2 S accumulation in a laboratory setting led to poor thiol formation, while natural yeast populations may pr oduce mor e sufficient H 2 S in response to nutritional stress (e.g.nitrogen limitation) (Henschke and Jiranek 1991, Jiranek et al. 1995, Bell and Henschke 2005 ).

C 6 precursor consumption during early fermentation
The consumption of unsaturated C 6 compounds, including ( E )hex-2-enal and ( E )-hex-2-en-1-ol, was investigated.Consistent with pr e vious r eports, the concentr ation of C 6 compounds significantl y decr eased in the presence of y east regar dless of strain tested (Fig. 3 ).An ANOVA sho w ed statistically significant variabil-ities in the residual concentrations of C 6 compounds among the 11 str ains, whic h may suggest differ ences in abilities to withstand α,β-unsaturated aldehyde toxicity (Kubo et al. 2003, Matsui 2006, Ma et al. 2019 ).High residual concentrations of C 6 compounds were detected in the fermentations pr oduced fr om F15, a commonly used commercial wine strain with high thiol output; M2 also sho w ed retar ded earl y-sta ge C 6 detoxification compared with labor atory str ains BY4743 or BY4741.Mor eov er, a time-course assay was performed to tr ac k ( E )-hex-2-enal concentration and its deri vati ves in the presence of BY4741 or BY4741-oye2 oye3 double deletant.In both cases, the concentration of ( E )-hex-2-enal decreased almost immediately after yeast inoculation and fell below the detection threshold within 10 h.Meanwhile, deri vati ves of ( E )hex-2-enal reduction accumulated in the fermentations (Fig. 4 ).Deletion of S. cerevisiae old y ello w enzymes ( OYE ) genes OYE2 and OYE3 , whic h hav e been shown to be r esponsible for the r eduction of α,β-unsaturated aldehydes (Williams et al. 2002, Trotter et al. 2006, Stuermer et al. 2007 ), had an insignificant impact on the elimination of ( E )-hex-2-enal, suggesting possible pathway redundancy.Unsaturated aldehydes pose significant cellular toxicity and are therefore considered to be an antimicrobial mechanism a gainst unwanted micr oor ganisms.In turn, winemaking yeasts are selected for unsaturated C 6 -resistance, particularly due to the high unsaturated C 6 , or "green leaf aldehydes" released from dama ged gr a pe tissue during harv est and mec hanical pr essing (Joslin et al. 1978, Hammerbacher et al. 2019 ).
Early kinetics of C 6 precursor consumption was also investigated in BY4741 and the BY4741-oye2 oye3 double deletant, with the exogenous addition of ( E )-hex-2-enal and H 2 S in the form of NaSH to ensure that 3SH would be produced (Fig. 4 ).C 6 concentration diminished rapidly in the presence of yeast cell inoculation.In fact, e v en brief contact of yeast inoculant with ( E )-hex-2enal-spiked YNB medium, follo w ed b y y east r emov al via centrifugation, was sufficient to almost halve the quantifiable ( E )-hex-2enal (second data point in Fig. 4 A), which was consistent with the observation of rapid ( E )-hex-2-enal elimination in previous reports (Joslin et al. 1978, Harsch et al. 2013 ).In fact, the precursor elimination occurred faster than pr e vious estimations, as onl y tr ace le v els of ( E )-hex-2-enal were quantified after 5-6 h.Together with the non-detection of thiol compounds in 12 h ferments, it therefor e r aises the question of the dir ect contribution of H 2 S-C 6 pathw ay, to w ar d total thiol output in finished wine (Schneider et al. 2006, Subileau et al. 2008 ).Inter estingl y, in the presence of an exogenous source of H 2 S, we were able to detect 3SH at 24 h both with BY4741 (701.2 ± 94.6 ng L −1 , mean ± SEM) and BY4741-oye2 oye3 (439.27 ± 25.7 ng L −1 , mean ± SEM) (Fig. 4 D).3SHA was not detected in either group.Although the o ye2 o ye3 double deletion a ppear ed to show r educed 3SH pr oduction after 24 h, ther e was no statistically significant effect observed ( P = 0.0557); howe v er, since this P -v alue was v ery close to the thr eshold set for significance (0.05), further investigation on the role of the OYE genes is warranted.It should be noted that the profile of H 2 S release fr om exogenous NaSH, whic h pr oduces an initial burst of H 2 S within a r elativ el y short time window, is dr asticall y differ ent fr om the slo w er and persistent endogenous H 2 S pr oduction fr om the yeast population (Harsch et al. 2013, Song et al. 2014 ).Further investigation using slow-releasing H 2 S donors may better mimic the accumulation and release of yeast endogenous H 2 S and its impact in the context of physiologically comparable kinetics (Kashfi andOlson 2013 , Song et al. 2014 ).
OYE genes have been suggested to catalyze the reduction of α, β-unsaturated aldehydes (W illiams and Bruce 2002 , Yuan et al. 2011 ).Yet, deletion of OYE2 and OYE3 did not significantly impact the capability of BY4741 strain to metabolize ( E )-hex-2-enal, suggesting redundancies or diversions in the detoxification pathway.Indeed, due to the large aldehyde reductase family in the S. cerevisiae genome, it is difficult to pinpoint the gene solely responsible for precursor metabolism.

Conclusion
The dual role of H 2 S in VSCs and thiol compound production during the early stage of fermentation was investigated.To our knowledge, this preliminary work addresses, for the first time, the fine landscape of sulfur volatile compounds in the transitional stage of early fermentation.The variability of early H 2 S potential among yeast strains was found to have a significant influence on the chemical profile of VSCs.In light of the non-detection of 3SH/3SHA and r a pid elimination of fr ee C 6 pr ecursors in earl y ferments, our data does not support the direct contribution of endogenous H 2 S to early thiol output via the addition to the ( E )hex-2-enal precursor in SGM.Ho w ever, upon addition of exogenous H 2 S, the ele v ated concentr ation of labile H 2 S r esults in the production of 3SH and 3SHA, suggesting that certain conditions must be met to reach sufficient H 2 S concentrations to obtain thiols from the ( E )-hex-2-enal pathwa y.T he development of efficient thiol quantification methods for the specific analysis of juice or juice-like substrates may assist in shedding further light on the early kinetics of thiol compounds during alcoholic fermentation.

Figure 1 .
Figure 1.Mean H 2 S accumulation of 11 laboratory reference and commercial S. cerevisiae strains in SGM during the initial 12 h of fermentation ( n = 5).Plot shows 12 h time-course measurements of Abs 663 /Abs 600 indicating MetB clearance normalized with biomass growth at 10 min intervals.Strain names are denoted at the top of each plot.

Figur e 3 .
Figur e 3. P ost-fermentation residual level of ( E )-hex-2-enal in fermentations.Data are presented as mean ± SEM.Means follo w ed b y a common letter are not significantly different by multiple comparison in post-hoc Tuk e y's HSD test at 0.05 level of significance.